Assessing the biological status of fish in a river receiving pulp and paper mill effluents.

This study compared the use of sentinel species- and community-based field approaches for assessing the biological status of fish living in a river receiving pulp and paper mill effluents. Three approaches were compared. Two approaches used sentinel species. One of these involved an internal/external examination of the fish that leads to the calculation of a fish health assessment index (HAI) and the other involved biochemical measurements of hepatic mixed function oxidase (MFO) activity and plasma steroid levels. The third approach characterized the fish community structure according to an index of biotic integrity (IBI). The comparison focused on how the methods respond to the hypothesis that recent process modifications/effluent treatment changes, resulting in demonstrable improvements in effluent quality, have beneficial effects on fish. Neither of the approaches using sentinel fish indicated clear mill-related influences either before or after the process modifications/effluent treatment changes. There was no evidence of depressed plasma steroids and increased MFO activity in fish frequently associated with mill effluent exposure in previous studies. While the HAI was higher at stations downstream from two mills, this could not be linked to effluent exposure alone. In contrast, the study of community structure showed a substantial improvement in fish assemblages at all the mill sites.

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer.

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer-based broad-spectrum immunotherapy of malignant disease.

Targeting p53, hdm2, and CD19: vaccination and immunologic strategies.

Peptides presented by class I major histocompatibility complex (MHC) molecules and derived from normal self-proteins that are expressed at elevated levels by cells from a variety of human (Hu) malignancies provide, in theory, potential target antigens for a broad-spectrum, cytotoxic T lymphocyte (CTL)-based immunotherapy of cancer and hematologic malignancies. However, as such tumor- and leukemia-associated self-proteins are also expressed at low levels in some types of normal tissues, such as thymus, spleen and lymphohemopoietic cells, these self-MHC-self-peptide complexes may also represent thymic and/or peripheral tolerogens, thereby preventing immune responses. This is particularly true for class I MHC-peptide complexes expressed by bone marrow-derived cells in the thymus, as such expression would cause negative selection of immature thymic T cells with high avidity for self-MHC-self-peptide complexes. This intrathymic deletion of potentially self-reactive T cells could result in a peripheral T cell repertoire purged of CTL precursors with sufficient avidity to recognize natural tumor associated self-epitopes presented by class I MHC molecules on tumor cells. HLA-transgenic (Tg) mice provide the basis of an experimental strategy that exploits species differences between Hu and murine (Mu) protein sequences in order to circumvent self-tolerance and obtain HLA-restricted CTL specific for epitopes derived from tumor- and leukemia-associated Hu self proteins, such as p53, Her-2/neu, hdm2 and CD19.

Engineering of a proteolytically stable human beta 2-adrenergic receptor/maltose-binding protein fusion and production of the chimeric protein in Escherichia coli and baculovirus-infected insect cells.

The hydrophobic human beta 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein (MalE) in Escherichia coli. Photoaffinity labeling with the adrenergic ligand [125I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli. The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other beta 2 adrenergic receptor constructs in E. coli. After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni2+ ions (binding to a C-terminal His6-tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli. The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.

Perspectives on Canadian field studies examining the potential of pulp and paper mill effluent to affect fish reproduction.

The results and interpretations of published Canadian field studies on the reproductive status of fish in waters receiving pulp and paper mill effluent discharges were reviewed. Most of the information was obtained from indicator measurements such as gonad size, fecundity, and serum steroid levels in wild fish sampled at reference and effluent-exposed sites. Difficulties in selecting appropriate sampling sites, natural variability, and the ecological relevance of the indicator measurements were identified as major complicating factors for the interpretation of the field data. Consequently, it was not possible to conclude to what extent, if any, widespread effects on fish reproduction are being caused by pulp and paper mill effluents or that specific manufacturing processes are causing such effects. Further research on the normal variability and predictive capability of reproductive indicators, for example, using an integrated approach (i.e., laboratory testing, mesocosm studies, and field work), is recommended.

Improved effluent quality at a bleached kraft mill as determined by laboratory biotests.

A life-cycle experiment with fathead minnows and Ceriodaphnia survival/reproduction tests were used to evaluate the quality of the effluent from a bleached kraft mill after the implementation of various process modifications and effluent treatment changes. In the life-cycle experiment, the fish were exposed in the laboratory to well water (control) and five concentrations (1.25%, 2.5%, 5%, 10%, or 20%) of effluent from the egg stage to sexual maturity and reproduction (approximately 190 d). None of the effluent concentrations significantly affected the hatching of the eggs, the mortality, weight, length, gender balance, reproduction, and prevalence of visible morphological or histopathological abnormalities of the hatched fish, and the hatchability of the first generation eggs. In Ceriodaphnia tests, the IC25 of the effluent affecting reproduction was approximately 80%. This threshold concentration is well above the 0.7% average yearly concentration of the effluent that exists in the recipient near the point of discharge. The results of these biotests were compared to the results of the same biotests conducted earlier with the effluent from the mill prior to process and treatment modifications. The comparison indicated that since the earlier work, the quality of the mill's effluent improved substantially. Threshold concentrations affecting fathead minnows in the life-cycle experiment and the Ceriodaphnia tests increased by more than eightfold and approximately twofold, respectively. While the most important change in the mill operating conditions responsible for the improvement could not be identified, these results indicate that mills can undertake process and treatment modifications that result in the discharge of effluents seemingly compatible with the aquatic environment.

Crystal structure of the bifunctional soybean Bowman-Birk inhibitor at 0.28-nm resolution. Structural peculiarities in a folded protein conformation.

The Bowman-Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin-reactive and chymotrypsin-reactive subdomains. In this report, the crystal structure of this anticarcinogenic protein has been determined to 0.28-nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemistry 31, 999-1010] in the relative orientation of the two enzyme-insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin-insertion loop, and in the orientation of several amino acid side chains. The proximity of Met27 and Gln48 in the X-ray structure contradicts the solution structure, in which these two side chains point away from each other. The significant effect of a Met27-->Ile replacement on the inhibitory activity of the chymotrypsin-reactive subdomain agrees with the X-ray structure. Exposed hydrophobic patches, the presence of charged amino acid residues, and the presence of water molecules in the protein interior are in contrast to standard proteins that comprise a hydrophobic core and exposed polar amino acids.

The effects of a secondary-treated bleached kraft mill effluent on aquatic organisms as assessed by short-term and long-term laboratory tests.

The chronic effects of secondary-treated effluent from a bleached kraft mill were assessed by means of long-term and short-term laboratory tests. In the long-term test, the effects of the effluent on the life cycle of fathead minnows (Pimephales promelas) were studied. In this experiment, which began with the egg stage and continued through to sexual maturity and reproduction, the fish were exposed in the laboratory to well water (control) and five concentrations (viz., 1.25, 2.5, 5, 10, or 20%) of effluent for 275 days. The effluent concentrations did not significantly affect the hatching of the eggs, the mortality of the hatched fish, the incidence of visible morphological abnormalities, the mortality and the hatchability of the first generation eggs and larvae, and the weights of minnows at various stages of development. Based on a conservative evaluation of the data, a significant finding of this work was that effluent concentrations > or = 2.5% caused lower egg production as well as changes in the gender balance (i.e., increased numbers of individuals with male secondary sexual characteristics) of the fish. Further work is required to understand the causes and ecological significance of these findings. Two short-term tests, each lasting 7 days, were also run. In one, even 100% effluent did not reduce the survival or growth of minnow larvae, correctly predicting the lack of effluent effects on similar endpoints in the long-term test. In the other short-term test, while the survival of Ceriodaphnia was also unaffected by 100% effluent, their reproductive capacity was reduced, but only at effluent concentrations an order of magnitude greater than those affecting the reproduction of minnows in the long-term test.

Effects of a secondary-treated thermomechanical pulp mill effluent on aquatic organisms as assessed by short- and long-term laboratory tests.

The chronic effects of secondary-treated effluent from a thermomechanical pulp (TMP) mill were assessed by means of long-term and short-term laboratory toxicity tests. The effluent used for the tests was sampled at a western Canadian mill using mostly softwoods and < 10% recycled fiber as furnish. In the long-term test, the effects of the effluent on the life cycle of fathead minnows (Pimephales promelas) were studied. In this experiment, which began with the egg stage and continued through to sexual maturity and reproduction, the fish were exposed in the laboratory to well water (control) and five concentrations (1.25%, 2.5%, 5%, 10%, or 20%) of effluent for 202 d. None of the effluent concentrations significantly affected the hatching of the eggs, the mortality, weight, length, gonad size, gender balance, and reproduction of the hatched fish, the prevalence of gross morphological and histopathological changes, and the hatchability of the first generation eggs. Two short-term tests, each lasting 7 d, were also performed. In these tests, 100% effluent caused no change in the survival/growth of minnow larvae or in the survival/reproduction of Ceriodaphnia.

A laboratory exposure procedure for screening pulp and paper mill effluents for the potential of causing increased mixed function oxidase activity in fish.

To better understand the relationships between pulp manufacturing processes and mixed function oxidase (MFO) enzyme induction in fish, a practical and standardized exposure procedure is required. This study was undertaken to develop a laboratory-based exposure procedure to quantify the relative MFO induction potencies of different types of pulp and paper mill effluents. One major consideration in developing the procedure was to ensure that the protocol was practical so that tests could be performed in a short time, with small volumes of effluents and using simple experimental conditions. A series of concentration-response and time-course experiments were conducted to find the minimum time and effluent concentration which could distinguish the ability of different effluents to cause significant MFO induction in rainbow trout in the laboratory. Experiments were also conducted to determine the effects of biotic and abiotic factors such as loading density, fish size and feeding regime. This study showed that the exposure of rainbow trout in the laboratory to 10% concentration of secondary-treated effluent for 96 h caused significant increases in hepatic MFO activity. The magnitude of MFO induction was comparable to other field and laboratory observations. While fish size, loading density and feeding regime were found to affect the test results, consistent responses within a laboratory using this protocol are possible, provided that these factors are standardized. Therefore, the short-term exposure approach described in this paper could be a relevant tool for assessing the ability of different types of pulp and paper mill effluents to cause MFO induction in fish.

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.